Parkinson Disease and -Synuclein

Transcription

1 Parkinson Disease and -Synuclein BIO200, WI13 In 1817, a British physician named James Parkinson carefully described a series of elderly patients with severe movement problems in a monograph Essay on the Shaking Palsy. Based on that first description, this disease became known as Parkinson disease (PD). In Parkinson s time, this disease was thought to be a form of madness as patients seemed to withdraw from the world. We now know that PD is a neurodegenerative disorder in which the brain s function slowly but irreversibly deteriorates. The incidence of PD increases with age; it is estimated that about 1% of Americans age 65 have PD but that the frequency increases to 5% at age 85. Therefore, PD is becoming much more common in the United States as our population is living longer. PD is a progressive disease, meaning that it starts out mildly but gets continually worse. PD patients are characterized by having a tremor in the hands when a person is at rest. Furthermore, PD patients often begin to show rigidity or slowness of movement or even freezing where they suddenly can t make voluntary movements. Overall, PD patients show difficulty in both starting and stopping voluntary movements. Even with the best medical care possible, most PD patients will experience profound impairment within 5-10 years of their diagnosis. This is a very scary disease. After death, the brains of PD patients all show a series of very similar hallmarks. Most importantly, many neurons in the substantia nigra pars compacta (SNpc) region of the brain will have died. These neurons express the enzyme tyrosine hydroxylase which catalyzed the hydroxylation of the amino acid tyrosine as one key step to making the neurotransmitter dopamine (right). Thus, these neurons are often referred to as the dopaminergic (DA) neurons. It is believed that the loss of dopamine production causes most of the movement problems in PD patients. Within the surviving neurons, large cytoplasmic inclusions are seen. These are known as Lewy Bodies are the only definitive means of diagnosing PD. What causes PD? In about 95% of the cases, we really don t know. These cases are referred to as idiopathic or sporadic cases. However, in about one patient in twenty, the disease is inherited. For the last 15 years, many PD researchers have worked to understand these inherited cases of PD. The hope is that once we understand the inherited disease, we will have some important insights into the much more common idiopathic cases. There are at least ten different inherited forms of PD. The most common inherited form of PD was the first one to be described in Scientists studied two large families in Greece and Sicily that showed a very high frequency of early-onset PD. By beginning with pedigree analysis and putting a gene mapping technology on it, they identified that PD can be inherited in an autosomal dominant fashion and that the responsible gene is SCNA. Other research groups found other SCNA mutations in unrelated families that also showed autosomal dominant, early-onset PD. The SCNA gene encodes a small protein of just 140 amino acids called -synuclein. Strikingly, at about that same time other research groups determined that the major protein found in Lewy Bodies of idiopathic PD cases is synuclein. The remarkable convergence of these two pieces of evidence seems to provide hope that we were beginning to understand PD. Once we understand PD, we may be able to cure PD.

2 There are several different mutations in -synuclein that are associated with inherited PD. Three different missense mutations [A30P (which means that an Alanine at amino acid #30 is replaced with a Proline), E46K and A53T] are associated with PD. In other families, a duplication or triplication of the wildtype SCNA gene is associated with PD. Unfortunately, none of these genetic modifications are found in idiopathic cases of PD. Notice that the last two paragraphs just say that SCNA mutations are associated with inherited PD. But do these mutations cause PD? Or are they just innocent bystanders? Or are they an effect of the disease that is caused by something else? The statistics told us that it was highly improbable that the SCNA mutations were present in the patients with inherited PD simply by chance, but the real proof came from animal studies. In general, animals don t get Parkinson disease. But if SCNA mutations or extra copies of the gene causes PD, then adding extra copies of the wildtype SCNA or the mutant SCNA genes should cause PD in animals. As predicted, adding SCNA genes to mice or rats causes neurological defects that look a lot like PD. We can even put the SCNA genes into flies, worms or even yeast and make them sick. Furthermore, these animals also show Lewy Bodies in the surviving SNpc neurons, just like human PD patients. This provided strong evidence that the -synuclein mutations really cause inherited PD and also provided strong evidence that the production of Lewy Bodies causes PD (rather than simply being one side effect of PD). Finally, these transgenic animals provide a good experimental system to study PD and to develop drugs to treat PD. So what is -synuclein and what does it normally do? Despite intensive study over the last 15 years, we still don t have a solid answer. We know that -synuclein is expressed in most cell types in humans but shows the highest levels of expression in the brain. It s a small protein of 140 amino acids that seems to have very little secondary and tertiary structure when found in the cytosol. At least part of the time, however, the protein isn t simply dissolved in the cytosol but rather is associated with a membrane. Part of the protein can form an amphipathic -helix that allows it to bind to fatty acids or even to bind one face of a phospholipid bilayer. Notice that this is not a transmembrane domain but simply a peripheral association. -synuclein has been reported to associate with many different membranes, from mitochondria to the plasma membrane to vesicles at the presynaptic terminals. (As you may know, neurons are connected together by synapses or small gaps across which they communicate. The presynaptic side is the cell that sends the message while the postsynaptic side is the cell that receives the message). What -synuclein is normally doing for these neurons remains unclear. Mice that are homozygously lacking the -synuclein gene seem to be just fine. When -synuclein is not associated with a membrane, other structures can sometimes form. The region of amino acids can form a -sheet which can then stick to another -sheet in another -synuclein molecule. This sticks to another and another and another, forming a homo-oligomer which is also known as a protofibril. The protofibrils can aggregate together to make the fibrils that seem to be the core of a Lewy Body. The three known mutations in -

3 synuclein that cause inherited PD (A30P, E46K and A53T) all increase the rate of fibril formation (although they do so in different ways). Furthermore, extra copies of the gene mean that more of the - synuclein protein is around which also facilitates fibril formation. Importantly, it seems that the presence of the neurotransmitter dopamine (remember that these are the dopaminergic neurons) also increases the rate of fibril formation. Why do Lewy Bodies lead to death of the DA neurons and PD? Surprisingly, that isn t entirely clear. It seems that the protein aggregations interfere with normal processes of breaking down and recycling proteins and will also decrease mitochondrial function. Aggregation of -synuclein to fibrils and Lewy bodies. Modified from Lashuel and Hirling, 2006 Therefore, it seems that Parkinson disease is really a problem of protein aggregation. If - synuclein aggregates into an insoluble Lewy Body, PD develops. Strikingly, other neurodegenerative disorders also seem to be caused by (or, at least, associated with) aberrant protein aggregation. Huntington s disease and Alzheimer s disease are two other progressive, neurodegenerative disorders. Like PD, both of these diseases are also characterized by neuron death and the surviving neurons show aggregations of different proteins. Understanding how to control protein aggregation in and around neurons could lead to revolutionary treatments for all three of these diseases. Optional background reading on Parkinson Disease and -synuclein: If you d like some additional background reading, I recommend the following recent review articles. Both are linked from the course website and the full text is freely available from on-campus. Of course, you may also want to look up things in your textbook or some of the other texts available in the laboratory. Auluck, P.K., G. Caraveo, and S. Lindquist Synuclein: Membrane interactions and toxicity in Parkinson s Disease. Annu. Rev. Cell Biol. 26: Marques, O., and T.F. Outeiro Alpha-synuclein: from secretion to dysfunction and death. Cell Death Dis. 3:e350.

4 The Assignments: The previous few pages present a brief review of what we know about PD and -synuclein today but this remains a very active area of research. More information is being published every week indeed, in the last year more than 500 papers about -synuclein were published. If you want to understand the latest advances in PD and -synuclein you need to be able to read and understand those research papers. That skill is one that we will practice this term in BIO200. As indicated above, discovering the normal function (or functions) of -synuclein remains an active area of research. The following three primary scientific papers can be obtained through the class website and present some evidence about -synuclein function. If you're going to print them, I strongly recommend using the pdf format which will look exactly the same as the paper did in the printed journal. In some cases, you may need to print parts of them in color or consult online supplemental online material located on the journal s website. Part A: Devi, L., V. Raghavendran, B.M. Prabhu, N.G. Avadhani, and H.K. Anandatheerthavarada Mitochondrial import and accumulation of -synuclein impair complex I in human dopaminergic neuronal cultures and Parkinson disease brain. J. Biol. Chem. 283: Part B: Gorbatyuk, O., S. Li, L.F. Sullivan, W. Chen, G. Kondrikova, F.P. Manfredsson, R.J. Mandel, and N. Muzyczka The phosphorylation state of Ser-129 in human -synuclein determines neurodegeneration in a rat model of Parkinson disease. Proc. Natl. Acad. Sci. USA. 105: Part C: Butler, E.K., A. Voigt, A.K. Lutz, J.P. Toegel, E. Gerhardt, P. Karsten, B. Falkenburger, A. Reinartz, K.F. Winklhofer, and J.B. Schulz. The mitochondrial chaperone protein TRAP1 mitigates -synuclein toxicity. PLoS Genet. 8:e Two or three figures (or parts of figures) from each paper are identified below for some in-depth investigation. Begin by reading the abstract and then skimming through the paper looking at section headings and figure captions to see some of the major conclusions. Then return to this assignment and look more carefully at the questions that I ve asked. Notice that you will not need to read every single word of any of these papers! Indeed, scientists rarely read all of a paper; rather we read the parts that specifically interest us. As you focus in on a few figures for in-depth analysis, add labels and notes to the figure to help you follow the authors' arguments. Limit your responses to no more than four 1.5- spaced pages each for Parts A and B and five pages for Part C. All work must be done independently. Please do ask your instructor for guidance and suggestions if you get stuck, but you may not ask another student (regardless if that student is currently in BIO200 or not). Obviously, you ll need to do some background reading to understand the problem and some of the techniques and terms. Some problems can be quickly solved by looking up terms in your textbook or doing a quick internet search to get a feeling for how an experiment was done or even reviewing the introductory information in this document. In some cases, you may need to take a quick look at some of the papers that are listed in the reference list of that paper. In writing your short papers, consider your target audience to be another BIO200 student, so be sure to define or explain things that you had to look up. All abbreviations need to be defined the first time you use them. Please write your short papers in paragraph form, not as a series of disconnected answers to my questions. Be sure that you understand

5 what you re saying using words or phrases that you don t fully understand can lead to you looking very foolish! As with any biology paper, do not quote the paper or any other source; re-state what you have learned in your own words. List the paper (and any other papers and textbooks but not websites that you looked up) at the end of your paper (please see the Biology Student Handbook for correct formatting of references). To help with the writing, I have included the questions from a previous term and example essays answering the questions on the course website. Part A As discussed in the introduction section to this assignment, it s not really clear why aggregation of the -synuclein protein leads to death of dopaminergic neurons. There have been many observations that suggest that mitochondrial function can be impaired with -synuclein aggregation but not a lot of details are currently available. To help better understand the affects that -synuclein has on oxidative phosphorylation, Hindupur Anandatheerthavarada s laboratory at the University of Pennsylvania carried out a series of experiments and published their results in the Journal of Biological Chemistry. Look at the experiments represented by Figures 4C and 4D. Dopaminergic neurons were cultured on a plate and were given additional DNA by a process called transfection. The cells either got the vector as a negative control, WT/Syn which is a gene encoding the wildtype (or unmutated) - synuclein, +33/Syn which is an -synuclein mutant that doesn t go to the mitochondria or A53T/Syn which is an -synuclein with a substitution mutation. In Figure 4C they are measuring Complex I activity in an enzyme assay. What is the chemical reaction that is being catalyzed? Are they measuring the reactant or the product? Are they measuring how its amount or concentration changes over time? Why did they divide the enzymatic activity by the mg of protein in the sample? Part of this assay includes adding rotenone. What is rotenone and why is it important to use it to get an accurate measurement of Complex I activity? What can you conclude about the effect of -synuclein on Complex I function? In Figure 4D, the authors are measuring the concentration of reactive oxygen species (ROS) in these cells in arbitrary fluorescence units. How do these data correlate to the data in Figure 4C? Does this correlation make sense or is it confusing? Figure 7 shows some of the most important data in the paper. They look at two different brain tissues from 22 humans who had recently died. 11 of these humans had late-onset, idiopathic Parkinson disease and 11 did not. What is the specific hypothesis being tested in Figures 7C and 7D? Was the hypothesis supported or refuted (be sure to refer to the data in your answer). Notice that the scales of axes of these graphs are very different what does that tell us about -synuclein and mitochondrial activity in humans? Part A is worth up to 20 points and is due in class on Friday, January 25 th.

6 Part B Like many human proteins, -synuclein can be phosphorylated on several sites including Serine To better understand the role of this post-translational modification in a living mammal, Gorbatyuk et al. undertook a series of experiments using rats. These scientists used a heavily modified virus to deliver the human -synuclein gene to the substantia nigra pars compacta (SNc) of rats. They either used wildtype -synuclein or a mutant that changes Ser-129 to either alanine or aspartate. Why did the scientists mutate Ser-129 to these two specific amino acids and not glycine, tryptophan or any other amino acid? (Hint: look at the structure of these amino acids in your textbook). Figure 1 is a lovely picture, but can be a little hard to understand. In Figure 1A, why are some cells red, yellow or green? Why are the authors particularly interested in TH-positive cells? What do the authors conclude from the data shown in Figure 1A? In Figure 1C, what is the source of the red and green colors? What important information is Figure 1C showing us? Figure 3 shows a large number of brain slices that have been stained in various ways. Based upon these images, the authors conclude that the S129A mutant -synuclein is more toxic than wildtype -synuclein to dopamine-secreting neurons in the SNc. Which two images are being compared to reach this conclusion? Similarly, they conclude that the wildtype -synuclein shows increasing toxicity to the dopamine-secreting neurons over time. Which three images are being compared to reach this conclusion? The authors conclude that phosphorylation of -synuclein on Ser-129 is protective and should help prevent Parkinson disease. Which one portion of one figure best supports this conclusion? (Be specific for example, don t say Figure 9 but rather say Figure 9D ). No experiment, however, is perfect. For example, one limitation of these observations is that they were made in rats and not humans. Name and briefly describe one other limitation to this conclusion. Part B is worth up to 30 points and is due in class Friday, February 15 th. Part C The previous two papers have looked at -synuclein in mammalian cells and rodents. But many experiments can be difficult to do with higher eukaryotes since they are relatively expensive to take care of properly and take a long time to reproduce. Thus, some investigators have turned to lower eukaryotes to use them as model organisms. Invertebrates like worms (Caenorhabditis elegans), flies (Drosophila melanogaster) and even non-animals like yeast (Saccharomyces cerevisiae) have been very valuable in understanding a variety of human diseases. None of these organisms normally get Parkinson disease or even have a -synuclein gene. But if human -synuclein is expressed, all of these organisms demonstrate pathology that is similar to Parkinson disease.

7 In this paper, the Schulz lab uses Drosophila as their model system. Expression of the A53T mutant of human -synuclein leads to a decrease in the amount of dopamine (DA) in the flies brains. The -synuclein gene is present in every cell in the flies bodies why is the protein only present in the dopaminergic neurons? The western blot in Figure 1A shows that when two copies of the -synuclein gene are present per cell, the flies express about twice as much protein. But how can we be certain that proteins from the same number of cells are present on the blot in each lane? Imagine introducing one copy of the human -synuclein gene with either the +33/ -synuclein truncation mutant that Devi et al. described or the S129D substitution mutant from Gorbatyuk et al. and measuring the amount of dopamine in the flies brains. For each of these two mutants, predict the phenotype and briefly explain your answer. They conducted a genetic screen (don t worry about the details as it s very complicated) and identified TRAP1 as a gene whose loss exacerbates the DA loss caused by the A53T -synuclein. The TRAP1 gene is widely conserved among animals. Identify the one part of one figure that has the data showing that introducing the human TRAP1 gene into these flies decreases the number of neurons that are killed by the A53T -synuclein mutant. Be sure to explain which two datapoints you are comparing to support this conclusion. These scientists began with flies but also looked at TRAP1 and -synuclein function in mammalian cells. In Figure 6A, they look at the phenotype of mitochondrial fragmentation in SH- SY5Y cells. What are these cells and why were they chosen for this experiment? Normal mitochondria in these cells are elongated tubes (as shown in the control image), but introduction of the A53T - synuclein causes the mitochondria to appear as shorter fragments (as shown in the A53T image). How are the mitochondria being visualized in this experiment? The authors added extra copies of either wildtype TRAP1 or a D158N mutant TRAP1. Why was this specific TRAP1 mutant chosen? Does the wildtype TRAP1 affect mitochondrial morphology when A53T -synuclein is added? Does it affect mitochondrial morphology without added -synuclein? Does the mutant TRAP1 have any effect with or without added A53T -synuclein? Is it possible that elevated TRAP1 levels simply leads to less - synuclein protein being present? Finally, consider all three papers that you have read. Do their arguments and ideas overlap at all or are they all touching on different aspects of -synuclein? Collectively, do they provide any insight or suggestions into the normal function of -synuclein? Based on what you now know, what new areas of -synuclein should be studied? The truly excellent student papers will draw connections between two or three of these published articles that will provide some additional insight into how -synuclein functions either in normal or diseased conditions. Part C is worth up to 40 points and is due in class Wednesday, March 6 th.

8 Plagiarism Obviously your short papers for this assignment will rely heavily on the three assigned journal articles. As you read those articles, notice that they never quote other scientists. Rather they briefly summarize their work and cite them. You will need to do the same thing in your papers: in other words, never quote any parts of these papers. Furthermore, as you are reviewing and summarizing the journal articles, you need to be careful not to lift sentences (or parts of sentences) from the journal article and directly use them in your paper. Changing a word or two or reorganizing a sentence is not a sufficient change. You need to read and understand the contents of the journal article and then present it in your own words. Please review the section on Plagiarism from the North Central College Guide to Writing, Documentation and Information Resources for some very specific information on plagiarism definitions. Here s an example that may help illustrate what is allowed and what constitutes plagiarism. The following example text that appeared in an original research article: Deletion of any one or all three of the CAC genes is not lethal, indicating that there must be other activities in S. cerevisiae capable of chromatin assembly. Perhaps this is crucial information that the reader of your paper needs to understand. If you write Loss of any one or all three of the CAC genes is not lethal, showing that there must be other activities in S. cerevisiae capable of chromatin assembly you are guilty of plagiarism, because you only changed two words to synonyms. Additionally, writing We know that there must be other activities in S. cerevisiae capable of chromatin assembly, because deletion of any one or all three of the CAC genes is not lethal, is also plagiarism as the two parts of the sentence were simply inverted. Notice that in both examples, you have only slightly modified the language, but the core idea is simply stolen intact. Rather, think about the core idea being communicated and write, Losing the function of either CAC1, CAC2 and/or CAC3 still leaves a viable yeast cell. Thus, we can conclude that there must be at least one other mechanism to assemble chromatin. Only in this last case, the writer has thought about the content of the journal article and is now explaining it in his/her own words. If you aren t clear on a sentence or a section of your paper, please ask your instructor for assistance. Plagiarism is a serious academic crime and will be dealt with severely in this course. Penalties will range from getting a zero on the assignment to expulsion from the College.